School of Physiology (ETDs)

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Start date: 2021End date: 2024

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    The association between adult attachment style and pain perception in a South African cohort
    (University of the Witwatersrand, Johannesburg, 2024) Stamp, Gabriella Elisabeth
    Our response to threats, including pain, is believed to be learnt during our early interpersonal connections and experiences. Interpersonal relationships can be measured through four adult attachment style classifications: Secure, Dismissing, Preoccupied and Fearful, with the latter three being collectively classified as Insecure attachment styles. Preliminary epidemiological evidence suggests that Insecure attachment styles are more prevalent in those with chronic pain, while experimental studies investigating the association between adult attachment and pain are inconclusive. In two separate investigations, the aims of my research were (i) to determine the association between adult attachment style and chronic pain prevalence and burden in a South African population, and (ii) to determine the association between the different adult attachment styles and measures of experimental pain as a way of assessing a potential mechanism for possible differences in pain perception between the attachment styles. In the first study, a nationwide online survey of a general South African population assessed adult attachment style (using The Experience in Close Relationships - Relationship Structures (ECR-RS) Questionnaire), prevalence of chronic pain and psychological factors that are typically associated with pain, including depression, anxiety and pain catastrophising. In participants who reported experiencing chronic pain, the association with attachment style and pain burden (pain sites, severity and interference, using the Brief Pain Inventory) was further investigated. A total of 2371 participants completed the survey, with the cohort being generally young in age (median age 23 years; IQR 20-28), well-educated and primarily female (74%), with predominantly a middle-to-high socioeconomic status. In this cohort, I found a higher-than-expected prevalence of chronic pain (27%); previously reported prevalence data in a South African population found the prevalence to be 18%. All three Insecure attachment styles were associated with increased chronic pain prevalence when compared to the Secure attachment style (Dismissing: 31%, Odds ratio [95%CI] = 1.38 [1.02-1.85], p=0.037; Preoccupied: 42%, Odds ratio [95%CI] = 2.26 [1.62-3.13], p<0.001; Fearful: 49%, Odds ratio [95%CI] = 2.95 [2.03-4.29], p<0.001). In participants with chronic pain, adult attachment style was not directly associated with the overall burden of chronic pain. Rather, my study found that pain catastrophising was the mediating factor between Insecure adult attachment styles and an increased burden of chronic pain. Female volunteers who had completed the survey were invited to participate in the second study, which involved in-person experimental procedures to evaluate the experience of thermal pain and mechanisms of pain analgesia using the Conditioned Pain Modulation (CPM) paradigm. In the 103 young (median age 21 years; IQR 20- 23) and well-educated (all completed at least secondary education) sample, no significant relationship was found between attachment style and heat pain threshold (t(54) = -0.45, p = 0.654), heat pain tolerance (t(47) = -1.16, p = 0.250), and intensity of heat pain (Estimate [95%CI] = -0.11 [-0.34-0.11], p-value = 0.330). Similarly, descending pain inhibition (assessed using CPM) was not associated with adult attachment style (t(59) = -0.97, p = 0.338). In these South African cohorts, adult attachment style directly associated with chronic pain prevalence, with remarkably more than double the chronic pain prevalence in Fearfully, compared to Securely, attached individuals. A possible mechanism underlying the association between insecure attachment style and high chronic pain prevalence may be differences in pain modulatory pathways, which was investigated through the CPM test paradigm in Part 2 of my research. Pain catastrophising mediated the relationships between attachment style and burden of pain, highlighting the role and impact of cognitive factors and the perception of threat on both attachment style and pain. Adult attachment style did not associate with perception of experimental pain, nor did it associate with mechanisms of pain inhibition. The data of the two research components of my thesis highlight the differences between chronic clinical pain and the once-off experience of experimental pain in a controlled and safe environment. The negative data in the experimental study may be explained by three main limitations: (i) The experimental protocol was not threatening enough and was unlikely to consistently activate the adult attachment system; (ii) individuals with high attachment anxiety and attachment avoidance dimension scores did not participate in the experimental study, which means the sample was biased and may not have shown any differences even if the protocol was threatening enough to activate the attachment system; (iii) the experimental pain protocol likely does not capture the threatening nature of chronic pain due to complex interactions of psychosocial factors that accompany chronic pain. These limitations are informative for future experimental pain studies, and I believe that CPM cannot, at this point, be ruled out as a mechanism for the increased chronic pain prevalence in insecurely compared to securely attached individuals. Moreover, pain catastrophising as a potential mechanism underlying the association between adult attachment style and the prevalence of chronic pain may also be a potential avenue for future studies to pursue.
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    The effect of circadian misalignment on cardiometabolic parameters in a rat model of estrogen deficiency
    (University of the Witwatersrand, Johannesburg, 2024) Nthlane, Refentshe Amandu’s; Michel, Frédéric
    Menopausal women engaging in shift work are at an increased risk of cardiometabolic disorders (CMD). The increased risk is due to the dual impact of menopause-associated estrogen withdrawal and the shiftwork-induced irregular light exposure, which disrupts circadian rhythms, leading to chronic circadian misalignment. However, the combination of circadian misalignment related to shift work and menopause on cardiometabolic health remains poorly understood. Therefore, the present study aimed to determine whether circadian misalignment worsens cardiometabolic parameters in estrogen-deficient female spontaneously hypertensive rats (SHR). Circadian misalignment was induced by a 10-week chronic phase shift (CPS) protocol, and estrogen deficiency was induced by ovariectomy. Thirty-six female SHR were ovariectomized or sham- operated 7 weeks after birth, and then subjected to a CPS or control lighting (ctr light) schedule (n=9 per group). Body mass, food and water intake, blood pressure and fasting blood glucose concentrations were measured throughout the 10-week protocol. An oral glucose tolerance test was performed 3 days before the end of the 10-week protocol, while ventricular systolic and diastolic function were assessed by echocardiography at the end of the 10-week protocol. Organ mass was measured, and LDL concentration was determined from collected serum with ELISA. Ovariectomized rats were heavier than sham-operated rats overtime (211 ± 38 vs. 169 ± 22g; p <0.0001). Food intake and organ masses were greater in ovariectomized rats compared to the sham-operated rats. When normalized to body mass, the food intake and organ masses were lower than in the sham-operated rats. Ovariectomized rats had greater left ventricular (LV) end-diastolic diameter (5.1 ± 0.4 vs. 4.6 ± 0.6mm; p = 0.03) and LV end-systolic diameter (3.0 ± 0.5 vs. 2.4 ± 0.4mm; p = 0.004) than sham-operated rats. The ovariectomized rats also had reduced endocardial fractional shortening (41 ± 8 vs. 49 ± 4%) and LV ejection fraction (71 ± 10 vs. 80 ± 4%) (both p = 0.007) than sham-operated rats. The cardiometabolic parameters measured were similar between the CPS and control lighting rats, except for a greater water intake overtime (CPS: 29 ± 7.1 vs. ctr light: 26 ± 3.2ml/day; p = 0.048) and a reduced liver mass (CPS: 7.2 ± 0.6 vs. ctr light: 7.6 ± 0.8g; p = 0.03) in CPS rats. When normalized to body mass, sham-operated rats had a greater water intake than the ovariectomized rats. No interaction between ovariectomy and CPS was demonstrated. In conclusion, estrogen deficiency impairs systolic function in female SHR. However, circadian misalignment does not worsen the cardiometabolic parameters in female SHR. Because of their genetic predispositions, female SHR may already have abnormal circadian rhythms, illustrating the complex and multifaceted circadian regulation within this rat model
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    The impact of altitude on the prevalence and characteristics of Restless Legs Syndrome
    (University of the Witwatersrand, Johannesburg, 2023) Munian, Pariska
    Restless Legs Syndrome (RLS) is a neurological sensory disorder, characterised by the irresistible urge to move due to unpleasant, deep-seated paresthesias in the legs. The urge to move usually occurs in the evening, when an individual is at rest and the sensations experienced are alleviated with movement. The prevalence of RLS in a general population ranges from 2.5 to 10%, from studies across the globe. Differences in RLS prevalence have been noted between different ethnic groups, with individuals of European ancestry exhibiting greater prevalence of RLS compared to individuals of Asian and African ancestry. The aetiology of RLS is unclear; however, there is evidence of central nervous system iron dysregulation. Low partial pressure of oxygen at high altitude, which is a large distance above sea level, may exacerbate iron dysregulation which may account for the greater prevalence of RLS at high compared to low altitude, which is an area at sea level. However, the impact of altitude on the prevalence of RLS requires further investigation and is the aim of this study. To investigate the effect of altitude on the prevalence and characteristics of RLS, a questionnaire was administered to the general South African population at two altitudes: low altitude (Durban, South Africa) and higher altitude, (1753m above sea level, Johannesburg, South Africa). The survey was completed by 1291 participants (416 at low altitude and 875 at higher altitude). Using an online questionnaire, data were collected on demographic characteristics (including age, sex and ethnicity), the Cambridge-Hopkins RLS questionnaire (to assess the presence/absence of RLS), self-reported iron deficiency, subjective measures of sleep, measures of daytime sleepiness (using the Epworth Sleepiness Scale) and levels of fatigue (using the Fatigue Assessment Scale). RLS was significantly more prevalent at the higher altitude (n = 69, 7.9%) compared to low altitude (n = 20, 4.8%), which may be due to an increase in iron dysregualtion at high altitude, resulting from the low partial pressure of oxygen. Factors associated with RLS also were exacerbated at higher altitude; these include increased RLS severity (p = 0.003), increased daytime sleepiness (p = 0.04) and decreased self-reported iron levels (p = 0.03) in individuals with RLS at higher altitude compared with low altitude. RLS was less prevalent in individuals with African ancestry than in those with European ancestry at the higher altitude (p = 0.0025). However, RLS was more prevalent in individuals with African ancestry than in those with Indian ancestry at low altitude (p = 0.0004). My data therefore support that altitude appears relevant to the pathophysiology of RLS, with high altitude presenting as a risk factor for RLS and exacerbating some characteristics of RLS
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    Investigating the toxic effects of nyaope on the brain, liver, and kidney of Sprague- Dawley rats
    (University of the Witwatersrand, Johannesburg, 2023) Mathiki, Khethani Thendo
    Background. Worldwide drug use is estimated to be 345 million, encompassing both synthetic and plant-based medications, as well as both legal and illicit opioids. The use of these drugs is on the rise and is becoming a growing concern for public health. Moreover, the emergence of heroin dominant street drugs like nyaope have gained popularity, especially in low- and middle- income countries such as South Africa. The use of nyaope exposes individuals to the risk of substance use disorders (SUDs) and the array of mental health issues, as evidenced by social and clinical studies. Nyaope, a relatively inexpensive illicit drug, is commonly found in townships and outskirts of inner cities which are predominantly saturated with African/Black ethnic communities. Its composition varies depending on the geographical area but primarily consists of ingredients like heroin, opioid derivatives, rat poison, antiretroviral drugs (ARVs), and other substances. Similar to other opioids, heroin exerts its effects on the central nervous system (CNS) by acting on opioid receptors in various brain regions, including the prefrontal cortex (PFC), nucleus accumbens, and amygdala. These regions play a critical role in regulating mood and cognitive functions. Additionally, opioids undergo processing in the liver before being excreted by the kidneys. Our work sought to evaluate the effects of acute toxicity from the unique street drug cocktail known as nyaope on the behaviour and molecular markers of the PFC, liver, and kidney in Sprague-Dawley rats, despite the abundance of material already available on opioid usage. Methods. Twenty-five Sprague-Dawley rats were sourced from the Wits Research Animal Facility following ethical clearance and habituated for a duration of ten weeks. A pilot study involving three of these rats was conducted to determine the appropriate exposure dose of nyaope. Following the pilot study, the remaining twenty-two rats were divided into two groups; nyaope-treated (n=11) and saline-treated (n=11). Nyaope-treated rats received a single dose of nyaope at 0.4 mg/kg/bw and were subsequently exposed to the Open Field Test (OFT), which assesses various behavioural indices, including locomotor activity, mood, and exploratory behaviour, using the AnyMaze video tracking system. The saline-treated rats received a single dose of physiological saline at 0.4 mg/kg/bw and underwent the same 30-minute exposure to the OFT. After this exposure, the animals were placed in their respective home cages and qualitatively observed for an additional 30 minutes. Following this observation period, the rats were anaesthetized with isoflurane and euthanized exactly one hour after exposure to nyaope or saline. Tissues from the brain, liver, and kidney were collected, and RT-PCR was conducted to assess toxicity markers, including genes that code for proteins involved in the processes of apoptosis (BAX and Bcl-2), autophagy (SQSTM1/p62), microglial repair (ANXA3), and inflammation (IL-6). In addition, plasma samples were collected and analysed using IDEXX catalyst technology to examine the plasma presence of liver toxicity markers; aspartate transferase and alanine transferase, along with the kidney toxicity marker; creatinine. Results. The qualitative findings indicated that rats treated with nyaope exhibited reduced grooming behaviour. Additionally, the nyaope-treated rats experienced a phase of heightened activity followed by extreme lethargy. In contrast, the saline-treated rats displayed consistent mobility and curious behaviour. Compared to the saline-treated rats, the nyaope-treated rats exhibited clinical signs such as tremors, a rigid tail, hypoxia, and increased diuretic behaviour. When observing the track plots of the nyaope-treated rats, they tended to favour the outer zone in a thigmotaxis pattern, with few bouts into the centre, while the saline-treated rats showed more uniform movement within the OFT apparatus. Quantitative behavioural data using the AnyMaze tracking system revealed that nyaope-treated rats had decreased locomotor activity. They covered less total distance during the test and travelled shorter distances within the centre zone compared to saline-treated rats. Nyaope-treated rats also had fewer mobility episodes and moved at slower speeds on average than the saline-treated rats. In terms of mood assessment, the nyaope-treated rats spent less time mobile overall, both in the outer and centre zones and engaged in significantly fewer grooming bouts compared to the saline-treated rats. In the assessment of exploratory behaviour, it was noted that nyaope-treated rats exhibited fewer instances of rearing, line crossing, and head entries into the centre than the saline-treated rats.Regarding the molecular assessment of the brain and kidney, there were no significant differences in the expression of molecular markers between the two groups, except for a decreased expression of Bcl-2 (p < 0.001) in the kidneys of nyaope-treated rats compared withthe saline-treated rats. Additionally, plasma expression levels of AST, ALT, and creatinine were similar between the two groups. Conclusion. These findings indicate that exposure to 0.4 mg/kg/bw of nyaope for one hour does result in behavioural changes, even though it does not immediately lead to acute molecular toxicity in the brain, liver and kidney. Conversely, nyaope exposure causes a reduction in mRNA expression of Bcl-2, suggesting that the drug induces cell and tissue damage in the kidney through apoptosis
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    The effects of acute LPS-induced inflammation on cardiac morphology, geometry andfunction in spontaneously hypertensive rats
    (University of the Witwatersrand, Johannesburg, 2024) Fako, Kealeboga Mme; Millen , Aletta; Michel, Frederic
    It has been established that systemic inflammation negatively impacts myocardial structure and function, especially in individuals with comorbidities such as hypertension. Acute exposure to lipopolysaccharide (LPS), resulting in acute high-grade inflammation, has been demonstrated to induce cardiomyocyte oedema and apoptosis in the short-term, resulting in left ventricular (LV) systolic and diastolic dysfunction. While exposure to LPS-induced inflammation causes LV dysfunction in the short-term, the long-term consequences of exposure to acute high-grade inflammation on the structure and function of the heart remain unclear. Therefore, the current study aimed to ascertain the immediate and long-term effects of a single exposure to LPS on the structure and function of the heart and its potential compounding effects in a hypertensive model. Wistar-Kyoto rats (WKY, n=36) and spontaneously hypertensive rats (SHR, n=38) were randomly divided into two groups per rat strain. The control groups (WKY- control and SHR-control) received one injection of saline (1 ml/kg, i.p.). The LPS groups (WKY-LPS and SHR-LPS) received one injection of LPS (1 mg/kg, i.p.). Animals were then terminated either 24 hours (WKY, n=11; SHR, n=16) or 6 weeks (WKY, n=25; SHR, n=22) after the saline or LPS injections. Prior to termination, conventional and speckle-tracking echocardiography were performed on all animals under anaesthesia to ascertain the effects of LPS on LV geometry, systolic and diastolic function. Following termination, heart tissues were removed and weighed prior to storage. Total collagen content in the left ventricle was determined using the Picrosirius red stain. A mixed model two-way analysis of variance (ANOVA) was used to ascertain differences in echocardiographic parameters, the inflammatory cytokine and fibrosis, followed by a Tukey’s post hoc test. Pearson’s correlation was used to determine associations between collagen volume and echocardiographic parameters. After 24 hours, LPS administration significantly increased interleukin (IL)- 1β concentrations in WKY-LPS (p = 0.02), and SHR-LPS (p = 0.03) groups compared to their respective control groups. LPS-induced inflammation resulted in impaired LV diastolic function as indicated by impaired LV relaxation (E/A, septal and average e’) in SHR-LPS compared to SHR-control (all p < 0.05). LV passive stiffness (e’/a’) increased significantly in WKY-LPS compared to WKY-control (p = 0.05). However, heart weight was significantly higher in SHR-LPS compared to WKY-LPS due to hypertension, not inflammation (p = 0.02). LPS-induced inflammation also significantly decreased LV systolic function in the short-term, as indicated by a reduced left ventricular outflow tract (LVOT) velocity time integral (VTI, p = 0.0004) and LVOT peak velocity (Vmax, p = 0.008) in SHR-LPS compared to SHR-control. Hypertension significantly decreased left ventricular ejection fraction (LVEF, p = 0.02) and endocardial fractional shortening (FSend, p =0.03), which are markers of global systolic function, in SHR-LPS compared to WKY- LPS. LVOT VTI (p = 0.02) and Vmax (p = 0.03) were significantly lower in the SHR-LPS compared to WKY-LPS in response to hypertension. LPS administration significantly reduced circumferential (p = 0.03) and longitudinal strain (p = 0.02), which are markers of early systolic dysfunction, in SHR- LPS compared to SHR-control. Hypertension significantly reduced circumferential (p= 0.0004) and longitudinal strain (p = 0.01), in SHR-control compared to WKY-control, and in SHR-LPS compared to WKY-LPS (both p < 0.0001). There were also reductions in circumferential strain rate in SHR-control compared to WKY-control (p = 0.01), and in both circumferential (p < 0.0001) and longitudinal strain rate (p = 0.0005), in SHR- LPS compared to WKY-LPS. In the animals that were terminated 6 weeks after LPS exposure, there were no differences in IL- 1β (all p > 0.05). LPS-induced inflammation had no effect on any of the LV diastolic or systolic function parameters in any of the groups (all p > 0.05). However, heart weight (p = 0.03) and normalised heart weight (p = 0.02) were significantly higher in SHR-control compared to WKY-control due to hypertension. Similarly, heart weight (p = 0.02) and normalised heart weight (p = 0.0006) were significantly higher in SHR-LPS compared to WKY-LPS in response to the effect of hypertension. Hypertension significantly impaired LV relaxation (reduced septal e’) in SHR-control compared to WKY-control (p = 0.04) and in SHR-LPS compared to WKY- LPS (p = 0.04). LPS-induced inflammation had no significant effects on LVOT VTI and LVOT Vmax (all p > 0.05). Hypertension significantly reduced LVEF (p = 0.03) and FSend (p = 0.04) in SHR-control compared to WKY-control, as well as LVOT VTI in SHR-control compared to WKY-control (p = 0.04). LPS administration had no significant consequences on circumferential and longitudinal strain as well as circumferential and longitudinal strain rate (all p > 0.05). Hypertension significantly decreased circumferential (p = 0.005) and longitudinal strain (p < 0.0001) in SHR-control compared to WKY-control, and longitudinal strain in SHR-LPS compared to WKY-LPS (p = 0.002). There were also reductions in circumferential (p = 0.01) and longitudinal strain rate (p < 0.0001) in SHR-control compared to WKY-control, and in longitudinal strain rate in SHR-LPS compared to WKY-LPS (p = 0.002) due to hypertension. In the short-term groups, inflammation was significantly associated with impaired LV relaxation and passive stiffness, while collagen volume was significantly associated with impaired LV relaxation and myocardial deformation. In the long-term groups, inflammation was associated with impaired LV relaxation, passive stiffness, myocardial deformation and collagen volume, while collagen volume was significantly associated with impaired LV relaxation. In conclusion, acute LPS-induced high-grade inflammation resulted in impaired LV diastolic and systolic function after 24 hours. These changes were worsened in the animals predisposed to hypertension. Although majority of the LV systolic and diastolic function variables were reversed after six weeks, alterations in morphological and myocardial deformation were not reversed. Therefore, a single dose of LPS administration may impact structural remodelling and myocardial strain in rats predisposed to hypertension in the long-term
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    Understanding the in vitro effect of TNF-α and hyperglycaemia on endothelial activation
    (University of the Witwatersrand, Johannesburg, 2023) Amoni, Joel Ikechukwu; Millen, A; Gunter, S
    Background. Inflammation is one of the main underlying mechanisms in the development of cardiovascular disease (CVD). Indeed, in diseases characterised by high-grade systemicinflammation and in comorbid conditions, such as diabetes mellitus, it has been suggested that inflammation, at least in part, contribute to the increased risk of CVD. One of the earliest signs of inflammation-induced CVD is endothelial dysfunction. However, whether inflammation promotes endothelial dysfunction via the same signalling pathways in different pathological conditions characterised by systemic inflammation is not well understood. Inflammation- induced aberrant expression of microRNA (miRNA), small non-coding RNAs that function to regulate gene expression post-transcriptionally, have been linked to impaired endothelial function. However, the mechanisms whereby miRNAs may mediate endothelial activation requires further investigation. Therefore, the aim of this study was to investigate the molecular mechanisms involved in the regulation of endothelial function in different models of inflammation. Methods. EA.hy926 immortalized endothelial cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) + HAM’s F12 nutrient mix supplemented with 10% foetal bovine serum (FBS). EA.hy926 cells were exposed to tumour necrosis factor-alpha (TNF-α) at a concentration of 10ng/ml for 24 hours to induce an inflammatory response while the controls were cells exposed to plain media for 24 hours. EA.hy926 cells were also exposed to either 5mM or 30mM glucose for 72 hours, as a model of glycemia-induced inflammation, while the control cells were exposed to plain media for 72 hours. Total RNA was extracted from the cell pellets and subsequently reverse transcribed to miRNA cDNA and mRNA cDNA. Quantitative real time PCR was used to determine the relative expression of interleukin-6 (IL-6), vascular cell adhesion molecule 1 (VCAM-1), miRNA-155-5p, endothelial nitric oxide synthase (eNOS) and superoxide dismutase 2 (SOD-2). Additionally, an ELISA assay was used to determine the ratio of phosphorylated p65/total p65 in cells exposed to TNF-α (10ng/ml for 24 hours), glucose (30mM for 72 hours) and plain media controls. Results. Compared to control cells, the relative mRNA expression of the inflammatory marker IL-6 was significantly increased in the cells exposed to TNF-α (p=0.002) and 5mM (p=0.002) and 30mM (p = 0.0001) glucose, respectively. In addition, the relative mRNA expression of VCAM-1 was increased in the cells exposed to TNF-α (p <0.0001) and 30mM glucose (p =0.03) when compared to their respective controls. Interestingly, miRNA-155-5p expression was also increased in the cells exposed to TNF-α (p= 0.04), 5 mM glucose (p = 0.007) and 30 mM glucose (p = 0.02). Exposure to TNF-α, and 5 mM and 30 mM glucose resulted in increased eNOS expression compared to the control cells (p = 0.04, p=0.002 and p = 0.0002, respectively). The ratio of phosphorylated-to-total NF-κB p65 were not different in either the TNF-α exposed or glucose exposed cells compared to control cells (all p>0.05). Conclusion. These findings suggest that TNF-α and hyperglycaemia exposure resulted in endothelial dysfunction. However, hyperglycaemia caused much greater oxidative stress (increased eNOS) most likely due to glucose scavenging of nitric oxide (NO). This suggests that the underlying mechanisms of endothelial dysfunction occurring due to hyperglycaemia and inflammation may be driven by different mechanisms. This study highlights the need for further investigation into the mechanisms whereby miRNA-155-5p regulate endothelial dysfunction
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    The effects of lycopene on bone, kidney and pancreatic health of growing Wistar rats fed an obesogenic diet
    (University of the Witwatersrand, Johannesburg, 2024) Gomotsegang, Motlhale
    Dietary fructose causes obesity, oxidative stress, and inflammation that compromise bone, kidney, and pancreatic health. Lycopene, a phytochemical, has antioxidant and anti- inflammatory properties. Its potential prophylactic effects on diet-induced bone, kidney and pancreatic derangements were evaluated in growing Wistar rats fed a high-fructose diet. Ninety-six 21-day old Wistar rat pups (48 females, 48 males) randomly allocated to treatment groups: I - standard rat chow (SRC) + plain drinking water (PDW) + plain gelatine cube (PGC), II - SRC + 20% fructose as drinking water (FDW) + PGC, III - SRC + FDW + 100mg/kg body mass fenofibrate per day (FF), IV - SRC + FDW + 30mg/kg body mass lycopene per day and V - SRC + FDW + 60mg/kg body mass lycopene per day and VI - SRC + FDW + 100mg/kg body mass lycopene per day were fed for 84 days. Body, kidneys, pancreata, femora and tibiae masses were determined. Kidney and pancreatic lipid content, pancreatic TBARS concentration, SOD and GPX1 activities and plasma CTX-1 and osteocalcin concentrations were measured. Femora and tibia, length, mid-shaft, sub-trochanteric medio-lateral, head and neck transverse, medio-lateral, and epicondylar diameters, maximum distal epiphyseal and proximal epiphyseal breadths and breaking strength were measured. Treatments had no effect on rats’ pancreata and males’ kidney masses (P>0.05). Fenofibrate increased the females’ kidney masses (P = 0.0056). Rats’ tibiae and females’ femora masses were similar (P>0.05). CTX-1, osteocalcin and TBARS concentrations and SOD and GPX1 activities were similar (P>0.05). Treatments did not affect the rats’ kidney and males’ pancreatic lipid contents (P>0.05). Dietary fructose increased females’ pancreatic lipid content (P = 0.0048). Lycopene prevented the fructose-induced increased pancreatic lipid content (P = 0.0048). Rats’ tibiae and femora breaking strength and femora mid-shaft diameter (MIDD) of males were similar (P>0.05). Dietary fructose reduced the females’ MIDD and males’ sub-trochanteric medio- lateral diameter (STMLD) (P>0.05). Lycopene prevented the dietary fructose-induced MIDD decrease in females (P = 0.0003). Low dose lycopene decreased (P = 0.0003) the females’ STMLD. Dietary fructose mediated sexually dimorphic effects and supplemental lycopene can protect females against fructose-induced pancreatic lipid accretion and MIDD reduction and males against STMLD reduction. In females low dose lycopene may increase the risk of femora fracturing through reduced STMLD
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    The Epidemiology of Menstrual Pain in a South African University Population
    (University of the Witwatersrand, Johannesburg, 2023) Futi, Benedicte Malonda; Iacovides, Stella; Scheuermaier, Karine
    Dysmenorrhoea, pain associated with menstruation, is a significant public health concern among young women of reproductive age. Identifying associated risk factors for the development of dysmenorrhoea is essential to minimize the impact of monthly menstrual pain on the daily functioning of these women, both in a personal and professional capacity. However, epidemiological data on the prevalence and associated risk factors for dysmenorrhoea in South Africa are scarce. This study aimed to determine the prevalence of dysmenorrhoea and its associated risk factors in a South African university student and staff population. An online survey was distributed to all 26 public universities across South Africa. The final sample comprised data from 7280 participants, and I found a high prevalence [76.7% (95% CI, 75.7-77.6)] of moderate-to-severe dysmenorrhoea among the respondents. Factors significantly associated with increase odds of experiencing moderate to severe dysmenorrhoea included: having heavy (adjusted OR = 2.749, 95% CI 2.208-3.421; p < 0.001) menstrual flow, having a positive family history of dysmenorrhoea (adjusted OR = 1.615, 95% CI 1.346-1.938; p < 0.001), always experiencing poorer subjective sleep quality [“often” (OR= 1.595, 95% CI 1.16-2.191; p=0.004), “sometimes” (OR= 1.523, 95% CI 1.22-1.902; p=0.0002) and “rarely” (OR=2.046, 95% CI 1.596-2.623; p<0.0001)], and scoring higher on the central sensitisation inventory total score (adjusted OR= 1.033, 95% CI 1.026-1.04; p < 0.001). On the other hand, factors significantly associated with decrease odds of experiencing moderate to severe dysmenorrhoea included: older age at the time of the study (adjusted OR= 0.982, 95% CI 0.967-0.998; p= 0.0285), older age at menarche (adjusted OR = 0.938, 95% CI 0.89-0.989; p= 0.0186), having been pregnant (adjusted OR = 0.757, 95% CI 0.605-0.946; p= 0.0145), lower BMI (adjusted OR = 0.986, 95% CI 0.972-1; p = 0.044), being of European ancestry (adjusted OR = 0.698, 95% CI 0.567-0.859; p = 0.007), and having light menstrual flow (adjusted OR= 0.473, 95% CI 0.373-0.6; p < 0.001). I also found a significant impact of dysmenorrhoea on daily life, with 51.6% of respondents reporting absenteeism from school or work during menses and 88.4% of the respondents requiring pharmacological treatments, such as contraceptive pills and nonsteroidal anti-inflammatory drugs (NSAIDs), to manage their menstrual pain. The study highlights the need for increased awareness, education, and effective interventions aimed at reducing the prevalence and impact of dysmenorrhoea on women's lives. The implications of both the increased central sensitisation (CS) and the sleep-pain reciprocal relationship suggest that they could potentially lead to the development of chronic pain conditions. Future research should further explore the interventions and management strategies that could improve sleep quality and prevent the onset of central sensitisation, thus reducing the risk of developing chronic pain conditions. The findings have important implications for the management of dysmenorrhoea that can improve women's quality of life and promote better health outcomes. These findings also point towards the need to educate women about the importance of seeking medical attention for dysmenorrhoea and the potential long-term implications of untreated dysmenorrhoea
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    MicroRNA expression and arterial function in type II diabetes mellitus
    (University of the Witwatersrand, Johannesburg, 2024) Goldfein, Batsheva; Millen, Aletta
    Background. Type II diabetes mellitus (T2DM) is a major health concern which significantly contributes to the global cardiovascular disease (CVD) burden. Arterial dysfunction is considered a subclinical marker of CVD and is associated with an increased risk of cardiovascular events. However, treatment outcomes for T2DM patients remain suboptimal, mainly due to a poor understanding and the lack of an early marker for the identification of subclinical CVD. Recently, microRNAs (miRNA), small, non-coding RNA molecules that regulate major signalling pathways through post-transcriptional modification, have been identified as possible epigenetic regulators in the development of many diseases. MiR-146a-5p, in particular, has received considerable attention as a biomarker associated with several disease states including inflammation, T2DM and CVD. However, studies surrounding the role of miR- 146a-5p in arterial function and subclinical CVD risk in diabetic populations have yielded contradictory results. Therefore, the aim of this study was to determine the role of miR-146a-5p expression in the development of arterial dysfunction in patients with T2DM. Methods. This case control study (n=118) included participants with a previous diagnosis of insulin resistance or T2DM (n=67), and a non-DM control group (n=51). Demographic characteristics and CVD risk factors were assessed using standard approaches. Arterial function was measured using applanation tonometry and SphygmoCor software. From the recorded radial and aortic waveforms, central systolic (cSBP) and pulse pressure (cPP), augmentation pressure (AP) and the forward (FWP) and reflected wave pressure (RWP) were derived using a generalised transfer function. The carotid-femoral pulse wave velocity (PWV) was measured as a marker of arterial stiffness. Using a fasting blood sample, serum concentrations of tumour necrosis factor α (TNFα) and matrix metalloproteinase 1 (MMP1) were quantified by ELISA. Real time quantitative PCR was used to determine the relative expression of miR-146a-5p using the comparative CT method relative to an endogenous control miRNA, miR-16-5p. Differences in anthropometric variables, miRNA expression and arterial function between the two groups were determined using unpaired t-tests or Mann Whitney U tests, as appropriate. Associations between miR-146a-5p expression and arterial function were determined using Pearson’s correlations. Participants were further stratified according to CVD risk using the Framingham risk score (FRS), and the associations with miRNA expression analysed using multivariate linear regression. Results. Participants with DM had significantly higher body mass index (p=0.002), triglyceride levels (p=0.004), and systolic blood pressure (p<0.001) than the control participants. Diabetic participants also had increased CVD risk compared to the control group, as assessed using FRS (P<0.001). Participants with DM also had significantly higher cSBP (p=0.003), mean arterial pressure (p<0.001), peripheral PP (p=0.04), FWP (p=0.045) and PWV (p=0.04). The relative expression of miR-146a-5p was significantly increased in the DM group compared to the control group (p=0.02). Across the study cohort, miR-146a-5p expression was significantly associated with waist-to- hip ratio (partial r=0.29, p = 0.002), triglyceride concentrations (partial r=0.2, p = 0.04), the atherogenic index (partial r=0.20, p=0.04) and TNFα concentrations (partial r=0.23, p=0.02), in age, sex and race adjusted analysis. In multivariate adjusted analysis, miR- 146a-5p expression was not associated with any of the arterial function variables (all p>0.05). However, when stratifying participants based on CVD risk, in those with a high risk for CVD (FRS≥20), miR-146a-5p was inversely associated with peripheral PP (Std β=-0.76, p=0.03, cPP (Std β=-0.76, p=0.01), cSBP (Std β=-0.38, p=0.02), FWP (Std β=-0.68, p=0.04) and RWP (Std β=-0.66, p=0.04). When MMP1 was included as a confounder, these associations were no longer significant. Conclusion. MiR-146a-5p expression was significantly higher in participants with T2DM compared to control participants and was significantly associated with traditional CVD risk factors and inflammation. MiR-146a-5p expression was not association with arterial function measures in the total population. Interestingly, in patients at a high risk for CVD, decreased expression of miR-146a-5p was associated with increased pressure pulsatility and wave reflection. These associations were lost when MMP1, a marker of arterial remodelling, was included as a confounding factor. These results suggest that miR-146a-5p may have a regulatory role in the development of arterial dysfunction through arterial remodelling in persons at high risk for CVD.
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    Neurophysiological effects of citalopram in a rodent model of ACTH-induced HPA axis dysregulation
    (University of the Witwatersrand, Johannesburg, 2024) Lubbe, Andréa; Baijnath, Sooraj
    Background. Major depressive disorder (MDD) is a debilitating disorder that affects approximately 300 million people worldwide. Despite multiple theories being implicated in the pathogenesis of MDD, most antidepressant drugs primarily target the monoaminergic theory of depression. Selective serotonin reuptake inhibitors (SSRIs) form a part of the most prescribed antidepressants that target the monoaminergic system. Following treatment with antidepressants, around 30% of patients with MDD continue to experience depressive symptoms, rendering them treatment resistant. The most widely accepted definition of treatment resistant depression is the resistance to at least two antidepressant regimens following the proper dose for an adequate amount of time. Treatment resistance in MDD has been attributed to dysregulation of the hypothalamic-pituitary-adrenal (HPA). HPA axis dysregulation leads to chronically high levels of glucocorticoids and failure of the regulatory negative feedback system. Consequently, there is a need for the development of novel drugs and validated animal models to study the antidepressive mechanism of these drugs. The aim of this study was to validate a rodent model of depression via chronic administration of adrenocorticotropic hormone (ACTH), and to investigate the effects of citalopram (a SSRI) on depressive-like symptoms using neurobehavioural tests and the regional brain expression of molecular markers associated with depression. Methods. Thirty-nine Sprague-Dawley rats (n=20 female; n=19 male) were randomly divided into a control group (n = 10 female, n= 9 male) and a depressed group (n=10 female; n=10 male). Rats in the control group received daily subcutaneous injections of saline (100μg/day) for two weeks. The depressed group received ACTH (100μg/day) subcutaneously daily for two weeks. Following the 2-week administration of ACTH or saline, all animals received citalopram (10mg/kg bw) for four weeks intraperitoneally, concurrently with ACTH or saline. The open-field test was performed to determine anxiety-like behaviour by assessing locomotor activity and time spent in central versus peripheral zones. The OFT was performed prior to the initiation of ACTH or saline administration. Following the 2-week administration of ACTH, the OFT was performed again to determine the effects of ACTH. The OFT was performed for a final time following citalopram treatment. The sucrose preference test (SPT) was performed to determine the presence of anhedonia. During the test, the rats were placed in the SPT apparatus with two water bottles for 12 hours. Water bottles were weighed before and after the test. After the four weeks of drug administration, rats were terminated by decapitation using a rodent guillotine. Brains were surgically removed, and the prefrontal cortex, striatum, hippocampus, and midbrain were surgically dissected and frozen. The RNA was isolated, and cDNA synthesised. Molecular gene expression analysis was performed for BDNF, CREB, TrkB, Akt, mTOR, and eEF2 using real-time polymerase chain reaction. Results. During the OFT, immobility increased in male and female saline + citalopram and ACTH + citalopram rats after 6 weeks (all p<0.05). During the SPT, females that received ACTH + citalopram had a higher preference for regular water consumption (p=0.004). In males, sucrose preference ratio was lower in ACTH + citalopram males than in saline + citalopram males (p=0.007). Regional mRNA expression of BDNF was significantly different between males and females (p<0.003). In the striatum, ACTH + citalopram females had higher expression of BDNF compared to ACTH + citalopram males (p=0.007). In the midbrain, ACTH +citalopram males had higher expression of BDNF compared to ACTH + citalopram females (p=0.01). Regional expression of mRNA of CREB resulted in a sex (p<0.0001) and treatment effect in the prefrontal cortex and midbrain (p<0.05). In the prefrontal cortex and midbrain, saline + citalopram and ACTH + citalopram males had higher mRNA expression of CREB than females (all p<0.001). There was a sex effect in the TrkB mRNA expression in the prefrontal cortex (p=0.005), as ACTH + citalopram females had a higher mRNA expression than ACTH + citalopram males (p=0.02). In the striatum, ACTH administration led to a higher TrkB mRNA expression in females compared to males (p=0.04). No significant differences were observed in mTOR mRNA expression between any of the groups (all p>0.05). In the midbrain, ACTH + citalopram females had a lower eEF2 mRNA expression than ACTH + citalopram males (p=0.02). Conclusion. This study suggest that male animals may exhibit greater susceptibility to neurobehavioural effects of ACTH, as oestrogen in females may be protective in regulating HPA axis function. Neurobehavioural changes in male rats may be linked to ACTH-induced effects on the dopamine system, affecting locomotor activity and reward processing. These results aid our understanding of the pathophysiology of depression related to HPA axis dysregulation. Nevertheless, the effects of ACTH induced dysregulation of the HPA axis on monoamine signalling, neuroinflammation, and GABA/glutamate neurotransmission, warrant further investigation